Uptake and degradation of insulin and α2-macroglobulin-trypsin complex in rat adipocytes. Evidence for different pathways

The cell association and degradation of insulin and α₂-macroglobulin-trypsin complex were measured in rat adipocytes with or without various inhibitors in the attempt to clarify whether the two ligands were taken up by the same or by different pathways. Several inhibitors, and particularly those of membrane traffic, lysosomal function and transglutaminase activity, affected the two ligands differently. Thus, chloroquine (100 μM) reduced both the uptake of α₂-macroglobulin · trypsin and its receptor-mediated degradation by about 70%. In contrast, the uptake of insulin was increased 2–3-times and the receptor-mediated degradation was only slightly reduced. Methylamine (10 mM) and ammonium chloride (10 mM) reduced degradation of α₂-macroglobulin · trypsin markedly without affecting that of insulin. Leupeptin (100 μM) increased uptake and reduced degradation of α₂-macroglobulin · trypsin without affecting insulin. Dansylcadaverine (500 μM) almost abolished uptake and degradation of α₂-macroglobulin · trypsin but had little effect on insulin. Moreover, uptake and degradation of α₂-macroglobulin · trypsin was much more sensitive than insulin to the action of metabolic inhibitors such as dinitrophenol and cyanide. The results show that the two ligands are taken up by functionally different systems. In addition, they support the hypothesis that lysosomes play a relatively minor role in the receptor-mediated degradation of insulin.     

Uptake of rat and human alpha 2-macroglobulin-trypsin complexes into rat and human cells

Uptake of rat and human alpha 2-macroglobulin-trypsin complexes was measured in rat hepatocytes, rat and human adipocytes and human fibroblasts. Uptake and degradation of 125I-labelled rat complex were about one-third of that of the human complex in the various isolated cell types. In rat hepatocytes, the apparent Km for cell association of the rat complex was about 16 nM as compared to about 6 nM for the human complex. The Vmax values were similar, about 1 X 10(4) molecules X cell-1 X min-1. Thus, rat alpha 2-macroglobulin (an acute-phase protein) complexed with trypsin follows the same pathways of uptake as the human homologue, although with a somewhat lower affinity for the uptake system.     

Separate intracellular pathways for insulin receptor recycling and insulin degradation in isolated rat adipocytes

To gain insight into the sequence of events that follow endocytotic uptake of insulin receptor complexes, we have examined the interrelationship between the degradative pathway of the insulin ligand and the recycling pathway of the insulin receptor. Tris(hydroxymethyl)aminomethane and other nonamphoteric amines were found to selectively impair insulin receptor recycling while leaving the insulin-degradative pathway intact. In contrast, low concentrations of the lysosomotropic agent chloroquine markedly inhibited intracellular insulin degradation but had little or no affect on the recycling of internalized receptors. Thus, we conclude: (1) that insulin dissociates from its receptor after endocytotic uptake and both receptor and ligand follow a separate intracellular pathway; and (2) that receptor recycling and insulin degradation can be selectively inhibited by Tris and chloroquine, respectively, highlighting the potential usefulness of these agents as intracellular probes in the study of receptor-ligand metabolism.     

Uptake of alpha 2-macroglobulin-trypsin complex by human placenta is mediated by a microvillous membrane receptor

The fate of native alpha 2-macroglobulin (alpha 2M) or its trypsin complex (alpha 2M-T) was studied in the isolated dually-perfused lobule of term human placenta. [125I]-alpha 2M added to the maternal circuit was unchanged during the course of the perfusion with minimal activity becoming associated with the placental tissue. Transfer of radioactivity into the fetal circulation accounted for only 0.07 per cent of the initial dose after 2 h. In contrast, [125I]-alpha 2M-T was rapidly taken up into the placental tissue (nearly 28 per cent of the initial dose during the 2-h perfusion) and breakdown products were released into both maternal and fetal circulations. At the end of 2 h, radioactivity levels on the fetal side were 13 times higher than those found with the native protein. These indications of a classical receptor-mediated uptake and breakdown pathway were confirmed in experiments in which the acidotrophic agent chloroquine was added to the maternal circuit prior to the alpha 2M-T. In the presence of chloroquine, tissue uptake was inhibited and the subsequent release of radioactive degradation products into the fetal circuit was similar to the levels seen with alpha 2M. Incubation of term trophoblast cells at 37 degrees C with [125I]-alpha 2M-T revealed over three-fold greater cell-associated activity than was found with the native protein. In another series of experiments, a purified microvillous membrane fraction was prepared from term placentae using buffers containing 1 mM iodoacetate. In the presence of this proteolytic enzyme inhibitor, binding studies showed a single class of low affinity receptors for the alpha 2M-T complex capable of binding 4.8 +/- 1.3 (SEM) micrograms of complex per mg of membrane protein. There was no binding of the native protein.     

Mechanisms of insulin degradation by isolated rat adipocytes

Summary We have examined some of the chemical and biological characteristics of the insulin-derived cell-associated radioactivity following incubation of isolated adipocytes with ¹²⁵I-insulin (10^–10 M) for one hour at 37 °C S ephadex G-50 chromatography of the cell-associated radioactivity demonstrated three peaks: peak I eluted with the void volume and consisted of large molecular weight material; peak II comigrated with 1251-insulin; and peak III consisted of small molecular weight degradation products (probably iodotyrosine). When the insulin peak (peak II) was divided into fourths, it was found that the binding and biologic activity of this material was not homogenous; thus, binding and biologic activity (relative to native insulin) fell markedly from the earliest to the latest eluting fractions of this peak. Furthermore, when the entire peak 11 material was applied to DEAE-Sephacel and eluted with a 0.01–0.2 M NaCl gradient, three distinct peaks were observed. These peaks were all 90% TCA precipitable, whereas the ability of the latter two eluting peaks to precipitate with anti-insulin antiserum was markedly reduced. When similar experiments were performed with chloroquine-treated cells, a large increase in cell-associated radioactivity was observed, and Sephadex G-50 chromatography demonstrated that this increase was entirely confined to peaks I and II. When the insulin peak (peak II) was divided into fourths, it was found that chloroquine markedly inhibited the decreased binding and biologic activity, from the earliest to the latest eluting fraction of this peak. Furthermore, when the peak II material (Sephadex G-50) from chloroquine-treated cells was chromatographed on DEAE-Sephacel, this material eluted in a single peak which was 95% TCA precipitable and 106% precipitable by anti-insulin antiserum. In conclusion, these studies demonstrate that: 1) intermediate insulin-derived products with reduced binding and biologic activity are generated in the process of cellular insulin degradation, and 2) the formation of these intermediate products is mediated by a chloroquine-sensitive pathway.     

Receptor-mediated degradation of insulin in isolated rat adipocytes. Formation of a degradation product slightly smaller than insulin

More than 90% of the radioactivity associated with isolated rat adipocytes incubated with [TyrA14-125I]monoiodoinsulin represented at steady state iodoinsulin possessing full binding affinity. In contrast, about half of the radioactivity dissociating from the cells was [125I]monoiodotyrosine. The other half was of a molecular size similar to that of iodoinsulin as judged from gel-filtration chromatography. However, the descending limb of the 'insulin' peak (i.e., the smaller molecules) possessed a reduced binding activity compared with native iodoinsulin, material from the ascending limb, or a similar fraction isolated from dissociation medium from IM-9 lymphocytes, a cell type devoid of receptor-mediated insulin degradation. The cells, thus, release an intermediary degradation product.     

Two pathways for insulin metabolism in adipocytes

Using selected conditions, the appropriate collagenase, albumin and cell treatment, a preparation of isolated adipocytes was developed with no extracellular insulin degrading activity. Cell mediated insulin degradation rates were 0.68%±0.05%/100 000 cell/h using trichloracetic acid precipitability as a measure. Chloroquine (CQ) increased cell-associated radioactivity and decreased degradation while dansylcadaverine (DC), PCMBS and bacitracin (BAC) decreased degradation with no effect on binding. Extraction and chromatography of the cell-associated radioactivity showed 3 peaks, a large molecular weight peak, a small molecular weight peak and an insulin-sized peak. CQ, DC and BAC all decreased the small molecular weight peak while CQ and DC also increased the peak of large molecular weight radioactivity. Cell mediated insulin degradation in the presence of combinations of inhibitors suggested two pathways in adipocytes, one affected by inhibitors of the insulin degrading enzyme (IDE) (bacitracin and PCMBS) and the other altered by cell processing inhibitors (DC, CQ and phenylarsenoxide). Chloroquine altered the pattern of the insulin-sized cell-associated HPLC assayed degradation products, further supporting two pathways of degradation; one a chloroquine-sensitive and one a chloroquine-insensitive pathway.     

The plasma clearance of human alpha 2-macroglobulin-trypsin complex in the rat is mainly accounted for by uptake into hepatocytes

Rats were given intravenous injections of 125I-labelled human alpha 2-macroglobulin X trypsin. The half-time of disappearance of radioactivity from arterial blood was 2 min. External counting showed that radioactivity in the liver was maximal by 10 min and then decreased slowly. 87% of the injected dose was recovered in the liver by 10 min. Light- and electron microscopic autoradiography carried out on samples of liver fixed with glutaraldehyde 3 min or 30 min after the injection showed that 85-90% of the grains were over the hepatocytes and 4-9% were over the Kupffer cells. Thus, uptake into hepatocytes, and not into Kupffer cells as believed previously, appears to account for the major part of the uptake of alpha 2-macroglobulin X trypsin by the liver and thereby for its rapid removal from the blood.     

Reaction of proteinases with alpha 2-macroglobulin: rapid-kinetic evidence for a conformational rearrangement of the initial alpha 2-macroglobulin-trypsin complex.

The kinetics of the reaction of trypsin with alpha 2M were examined under pseudo-first-order conditions with excess inhibitor. Initial studies indicated that the fluorescent dye TNS is a suitable probe for monitoring the reaction over a wide concentration range of reactants. Titration experiments showed that the conformational changes associated with the binding of trypsin to alpha 2M result in an increased affinity of the inhibitor for TNS. Two distinct phases were observed when this dye was used to monitor the progress of the reaction. Approximately half of the fluorescence signal was generated during a rapid phase, with the remainder generated during a second, slower phase. The observed pseudo-first-order rate constant of the first phase varied linearly with the concentration of alpha 2M up to the highest concentration of inhibitor used, whereas the rate constant of the second phase was independent of alpha 2M concentration. The data fit a mechanism in which the association of trypsin with alpha 2M occurs in two consecutive, essentially irreversible steps, both leading to alterations in TNS fluorescence. The initial association occurs with a second-order rate constant of (1.0 +/- 0.1) X 10(7) M-1 s-1 and is followed by a slower, intramolecular conformational rearrangement of the initial complex with a rate constant of 1.4 +/- 0.2 s-1. The data are consistent with a previously proposed model for the reaction of proteinases with alpha 2M [Larsson et al. (1989) Biochemistry 28, 7636-7643].2+ this model, once an initial 1:1 alpha 2M-proteinase     

Evidence for surface glycoprotein involvement in the intracellular bioactivity of insulin in rat adipocytes.

Lectins specific for D-mannose (concanavalin A), N-acetyl-D-glucosamine (wheat-germ agglutinin) or D-galactose (Ricinus communis agglutinin I) inhibited insulin binding and activated glucose transport in rat adipocytes [Cherqui, Caron, Capeau & Picard (1982) Mol. Cell. Endocrinol. 28, 627-643]. In the present investigation, the intracellular activities of insulin and lectins on lipogenesis and protein synthesis were studied under conditions where neither agent had an effect on membrane transport processes. (1) When glucose transport was rate-limiting (0.5 mM-glucose), insulin (0.8 ng/ml) and lectins (20 micrograms/ml) increased lipogenesis by 2.4-3-fold. (2) When passive diffusion of glucose was amplified (10 mM-glucose), insulin (0.8 ng/ml) and lectins (20 micrograms/ml) increased lipogenesis by 1.6-1.8-fold even in the presence of 50 microM-cytochalasin B, which completely blocked glucose transport. (3) Insulin (6 ng/ml), concanavalin A and wheat-germ agglutinin (40 micrograms/ml) stimulated the incorporation of L-[U-14C]leucine into fat-cell protein 1.5-fold but did not modify alpha-aminoisobutyric acid uptake or 14C-labelled protein degradation. (4) Peanut and soya-bean agglutinins (specific for O-glycosidically-linked oligosaccharides), known not to alter insulin binding, were ineffective. (5) Lectin effects were dose-dependent and were markedly inhibited by specific monosaccharides (50 mM). (6) Insulin and lectin maximal effects were not additive and were completely abolished by neuraminidase treatment of fat-cells (0.05 unit/ml). These data indicate involvement of surface sialylated glycoproteins of the complex N-linked type in the insulin stimulation of glucose and amino acid intracellular metabolic processes. They suggest, together with our previous results, that the transmission of the insulin signal for both membrane and intracellular effects occurs via glycosylated effector entities of, or closely linked to, the insulin-receptor complex.     

Amiloride inhibits insulin sensitivity and responsiveness in rat adipocytes through different mechanisms.

To investigate the mechanisms by which amiloride inhibits insulin action rat adipocytes were treated with insulin and with amiloride added before or after energy depleting the cells with 2 mM KCN. Amiloride decreased the insulin response on 3-0-methylglucose transport, IGF-II- and insulin binding in both intact and energy depleted cells. In contrast, the sensitivity to insulin was inhibited by amiloride only when it was added before KCN. The effect of amiloride on insulin sensitivity was probably exerted through the impaired activation of the insulin receptor tyrosine kinase and the decreased insulin binding. However, insulin responsiveness was probably impaired through a direct effect on the plasma membrane proteins. In contrast to a recent report with pituitary cells, amiloride did not affect the activation of the inhibitory GTP-binding protein (Gi) in rat adipocytes.     

Receptor-mediated degradation of insulin in isolated rat adipocytes: Formation of a degradation product slightly smaller than insulin

More than 90% of the radioactivity associated with isolated rat adipocytes incubated with [Tyr^A14-¹²⁵I]monoiodoinsulin represented at steady state iodoinsulin possessing full binding affinity. In contrast, about half of the radioactivity dissociating from the cells was [¹²⁵I]monoiodotyrosine. The other half was of a molecular size similar to that of iodoinsulin as judged from gel-filtration chromatography. However, the descending limb of the ‘insulin’ peak (i.e., the smaller molecules) possessed a reduced binding activity compared with native iodoinsulin, material from the ascending limb, or a similar fraction isolated from dissociation medium from IM-9 lymphocytes, a cell type devoid of receptor-mediated insulin degradation. The cells, thus, release an intermediary degradation product.     

The cellular dynamics of hepatic receptors for alpha 2-macroglobulin.protease complex and for insulin are different

Making freshly isolated rat hepatocytes permeable by 0.4 g/liter digitonin doubled the number of binding sites for alpha 2-macroglobulin.trypsin complex without changing the affinity. Thus, digitonin unmasked a receptor pool, probably of intracellular origin. The total cellular binding capacity was measured in the presence of digitonin, the surface-exposed in its absence. Upon preincubation of the cells at 37 degrees C, the total cellular binding capacity for alpha 2-macroglobulin.trypsin decreased over a 2-h period to 0.26 of the initial value. By contrast, the surface-exposed binding capacity initially increased in response to a preincubation at 37 degrees C, reached after 20 min a peak value 1.74 times that at 0 time, followed by a decrease. Neither the increase in nor the loss of surface-exposed binding capacity was influenced by inhibitors of lysosomal functions, protein synthesis and glycosylation. Colchicine abolished the increase in surface-exposed binding capacity but not the disappearance. By contrast, phenylarsine oxide (inhibitor of endocytosis), N-ethylmaleimide, and phenylmethanesulphonyl fluoride inhibited the receptor loss, suggesting that the loss occurred by proteolysis. The insulin receptor concentration, studied in parallel, remained practically constant in the investigated period in the presence and absence of digitonin. Thus, the hepatic receptor for alpha 2-macroglobulin.protease complexes is regulated independently of other specialized plasma membrane proteins.     

Activation of glycogen synthase by insulin in rat adipocytes. Evidence of hormonal stimulation of multisite dephosphorylation by glucose transport-dependent and -independent pathways

Adipocytes were incubated with [32P]phosphate to achieve steady state labeling of glycogen synthase. The enzyme was then rapidly immunoprecipitated and subjected to electrophoresis on polyacrylamide slab gels in the presence of sodium dodecyl sulfate. The 32P-labeled glycogen synthase had an apparent molecular weight ( Mapp ) equal to 90,000. All of the [32P]phosphate could be recovered in two cyanogen bromide fragments. The larger fragment, CB-2 ( Mapp = 28,000), contained about five times more [32P]phosphate than the smaller fragment, CB-1 ( Mapp = 15,500). Insulin increased the activity ratio (-glucose-6-P/+glucose-6-P) of glycogen synthase from 0.12 to 0.26, but did not decrease the amount of [32P]phosphate in the enzyme. However, insulin promoted the formation of species of CB-2 of lower Mapp , suggesting dephosphorylation of sites that affected the electrophoretic mobility of the fragment. Glucose did not affect the mobility of CB-2, but slightly increased the activity ratio and decreased the [32P] phosphate by approximately 20%. With insulin plus glucose, the increase in activity ratio was much greater than the additive effects of either agent alone. The combination decreased the [32P]phosphate in each cyanogen bromide fragment by approximately 60%, indicating that the synergistic activation was due to enhanced dephosphorylation of multiple sites. 2-Deoxyglucose also promoted dephosphorylation of glycogen synthase, decreasing the 32P content of CB-1 and CB-2 by approximately 40% each. 3-O-Methylglucose was without effect. The results presented suggest that the activation of glycogen synthase by insulin via a glucose transport-dependent pathway may involve increased intracellular glucose-6-P which promotes dephosphorylation of sites in both CB-1 and CB-2. Activation by a glucose transport-independent pathway appears to be confined to sites located in CB-2.     

Lysosomal and non-lysosomal pathways of intracellular insulin degradation in isolated rat hepatocytes

The amount of 125I-insulin associated with freshly isolated hepatocytes was increased 50% in the presence of 0.2 mM chloroquine (CQ) after 2 h of incubation. The degradation of insulin by the hepatocytes incubated with CQ was significantly diminished as compared with control cells. Hepatocytes incubated with 125I-insulin in the presence of CQ showed a slower rate of ligand dissociation than control cells. More TCA-precipitable and less TCA-soluble material appeared in the dissociation buffer of CQ-treated cells. However, CQ inhibited only 25-35% of intracellular insulin degradation. Non-lysosomal intracellular insulin degradation appears to be responsible for the remaining portion of the ligand degradation by isolated hepatocytes.     

Internalization and degradation of a low affinity insulin ([LeuB24]insulin) by rat adipocytes

In Halobacterium halobium tactic responses towards light and chemoeffectors are accompanied by changes in the methylation level of methyl-accepting chemotaxis proteins (MCP). Taxis towards green light absorbed by the bacteriorhodopsin proton pump appears to be governed by Δ$\text{μ}$_H⁺-sensing. The addition of CCCP, an uncoupler, prevented the increase of MCP methylation in response to green light illumination, but had no effect on CH₃-incorporation followed by the addition of the attractants glucose, leucine and histidine. Similarly, CCCP did not change MCP demethylation in response to blue light illumination, a repelling stimuli.The sensitivity to an uncoupler of methylation linked to Δ$\text{μ}$_H⁺-mediated green light taxis is to be expected, while the independence of demethylation caused by the blue light of CCCP is an indication that in the latter case a specific photoreceptor governs phototaxis. Informed processing from the blue light receptor to MCP does not involve a change in the membrane potential.     

Insulin and alpha 2-macroglobulin-methylamine undergo endocytosis by different mechanisms in rat adipocytes: II. Comparison of intracellular events

A previous ultrastructural study showed that gold-labeled insulin (Au-I) and the non-hormonal ligand gold-labeled alpha-2-macroglobulin-methylamine (Au-alpha 2MGMA) underwent endocytosis by dissimilar cell surface structures on rat adipocytes. The present ultrastructural study compared the intracellular routes taken by these two ligands in adipocytes. Intracellular Au-alpha 2MGMA was initially found within apparent coated vesicles but Au-I was not, consistent with the previous demonstration that Au-alpha 2MGMA underwent endocytosis by coated pits whereas Au-I was internalized by uncoated micropinocytotic invaginations. Early in the endocytic pathway, the two ligands were segregated within separate small vesicles and tubulovesicles. Au-alpha 2MGMA was concentrated in a small number of these structures whereas Au-I was sparsely distributed among a relatively large number. Subsequently, the two endocytic pathways converged as the ligands intermingled within pale multivesicular bodies and lysosome-like structures. Au-I was less efficiently transferred to lysosomes than Au-alpha 2MGMA since a greater proportion of intracellular Au-I remained associated with small vesicles and tubulovesicles. This study indicates that early intracellular events in the endocytic pathways of insulin and alpha 2MGMA are distinct. These findings are discussed in light of the fundamentally dissimilar biological roles of these two molecules and the possible involvement of the endocytic pathway in the insulin signaling mechanism.     

Colloidal gold-labeled insulin complex. Characterization and binding to adipocytes

Biologically active insulin gold complex was used as an ultrastructural marker to study insulin binding sites, uptake, and internalization in isolated rat adipocytes. The preparation conditions for monodispersed particles, ca. 16 nm in diameter and loaded with approximately 100 insulin molecules, are reported. The complex is stable for at least six weeks. Single particles or small clusters were scattered across the cell membrane. The distribution of unbound receptors seemed to be independent of the extensive system of pre-existing surface connected vesicles in adipocytes. The uptake of particles took place predominantly via non-coated pinocytotic invaginations; clathrin-coated pits did not seem to be important for this process. Lysosome-like structures contained aggregates of 10-15 particles. These data suggest that insulin gold complex is a useful marker for the specific labeling of insulin binding sites.